Previously, a functional relationship was identified between RNA silencing and the gypsy insulator of Drosophila melanogaster. The activity of the gypsy insulator is decreased in piwi and aubergine Argonaute mutants but is improved when levels of the Rm62, SpindleE, or Armitage helicases are reduced. Both Rm62 and Piwi interact physically with the insulator protein CP190, and the Rm62 interaction is RNA dependent. Mutation of piwi, aubergine, Rm62, or armitage alters gypsy insulator body nuclear organization, consistent with observed effects on insulator function. Therefore, RNA silencing pathways may promote the multimerization of insulator complexes and/or their association with a nuclear scaffold. Recent studies have shown that CP190 plays a role in another distinct chromatin insulator complex, associating physically and functioning in concert with the dCTCF insulator protein at the Fab-8 insulator. Like the gypsy insulator, Fab-8 is not affected by mutations in the miRNA pathway and decreased in activity in piwi and aubergine mutants. However, Argonaute 2 (AGO2) mutants reduce Fab-8 but not gypsy insulator activity while Rm62 mutants have no effect on Fab-8 insulator function, suggesting that distinct RNA silencing mechanisms affect these two insulator complexes. Additionally, Ago2 associates physically with CP190, and this interaction is not RNA-dependent. Current efforts are focused on gaining mechanistic insight into AGO2 influence on Fab-8. Recent studies suggest that Drosophila CP190-containing chromatin insulator complexes such as gypsy and Fab-8 may harbor an additional RNA component. Isolated CP190 complexes show enrichment of RNA species of approximately 35 and 55nt. Deep sequencing of these RNAs and subsequent RT-PCR analysis indicates the presence of larger RNAs derived specifically from at least three genes. In addition, nucleases associate physically with CP190, and purified CP190 complexes are capable of degrading an artificial RNA substrate. Importantly, mutant flies heterozygous for deletions in certain nucleases display in vivo defects in Fab-8 but not gypsy insulator activity compared to wildtype. In these mutants, intergenic transcription in the vicinity of Fab-8 is decreased compared to wild type, and we speculate that association of nucleases with chromatin insulator complexes may reduce transcription of competitive transcripts that interfere with local enhancer activation.